Molecular Tension Probe for In Vitro Bioassays
Molecular Tension Probe for In Vitro Bioassays
复制标题
用于体外生物测定的分子张力探针
DOI：
10.1007/978-1-0716-2453-1_7
复制
发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Mikio Tanabe
中科院分区：
文献类型：
--
作者：
Sung-Bae Kim;Rika Fujii;Simon Miller;Mikio Tanabe
与基于细胞的生物测定法（cbb）相比，无细胞生物测定法（cfb）有其独特的优点，包括(i)快速和现场适用性，（ii）长期实用性，以及（iii）生物分析的通用性。作者先前介绍了一种独特的生物发光成像探针，用于照亮感兴趣的蛋白质-蛋白质相互作用（PPIs）所附加的分子张力。在本章中，我们举例说明了一个全长的人工荧光素酶通过最小的柔性连接体FRB- a23 -FKBP夹在FRB （fkbp12 -雷帕霉素相关蛋白的FKBP-雷帕霉素结合域）和FKBP （fk506结合蛋白）之间。rapamycin激活的PPIs在FRB和FKBP之间给夹在中间的荧光素酶增加了分子张力，定量地增强了酶的活性。融合蛋白FRB-A23-FKBP经过三步色谱柱纯化，在不同的循环流化床条件下具有生物分析功能。本章指导FRB-A23-FKBP从纯化到实际生物测定的详细方案。
Cell-free bioassays (CFBs) provide their own distinctive merits over cell-based bioassays (CBBs) including (i) rapid and on-site applicability, (ii) long-term utility, and (iii) bioanalytical versatility. The authors previously introduced a unique bioluminescent imaging probe for illuminating molecular tension appended by protein-protein interactions (PPIs) of interest. In this chapter, we exemplify that a full-length artificial luciferase is sandwiched between FRB (FKBP-rapamycin-binding domain of FKBP12-rapamycin-associated protein) and FKBP (FK506-binding protein) via minimal flexible linkers, named FRB-A23-FKBP. The rapamycin-activated PPIs between FRB and FKBP append molecular tension to the sandwiched luciferase, enhancing the enzymatic activity in a quantitative manner. The fusion protein, FRB-A23-FKBP, is three-step column-purified and the bioanalytical utility is characterized in various CFB conditions. This chapter guides the detailed protocols from the purification to the practical bioassays of FRB-A23-FKBP.